Cohort profile: Biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer recurrence and survival – the FOCUS Consortium

Purpose The overarching goal of the FOCUS (biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer (CRC) recurrence and survival) Consortium is to unravel the effect of folate and folate-mediated one-carbon metabolism (FOCM) biomarkers on CRC prognosis to provide clinically relevant advice on folate intake to cancer patients and define future tertiary prevention strategies. Participants The FOCUS Consortium is an international, prospective cohort of 2401 women and men above 18 years of age who were diagnosed with a primary invasive non-metastatic (stages I–III) CRC. The consortium comprises patients from Austria, two sites from the Netherlands, Germany and two sites from the USA. Patients are recruited after CRC diagnosis and followed at 6 and 12 months after enrolment. At each time point, sociodemographic data, data on health behaviour and clinical data are collected, blood samples are drawn. Findings to date An increased risk of cancer recurrences was observed among patients with higher compared with lower circulating folic acid concentrations. Furthermore, specific folate species within the FOCM pathway were associated with both inflammation and angiogenesis pathways among patients with CRC. In addition, higher vitamin B6 status was associated with better quality of life at 6 months post-treatment. Future plans Better insights into the research on associations between folate and FOCM biomarkers and clinical outcomes in patients with CRC will facilitate the development of guidelines regarding folate intake in order to provide clinically relevant advice to patients with cancer, health professionals involved in patient care, and ultimately further tertiary prevention strategies in the future. The FOCUS Consortium offers an excellent infrastructure for short-term and long-term research projects and for combining additional biomarkers and data resulting from the individual cohorts within the next years, for example, microbiome data, omics and multiomics data or CT-quantified body composition data.


INTRODUCTION
Among men and women worldwide, colorectal cancer (CRC) accounts for nearly 10% of all incident cancer cases. 1 In Europe, 5-year survival for patients with CRC is approximately 55%, with substantial differences by stage. 2 3 The number of patients with CRC continues to increase due to implementation of improved screening strategies and/or enhanced treatment modalities. 4

STRENGTHS AND LIMITATIONS OF THIS STUDY
⇒ FOCUS is the largest consortium to date addressing the research question of folate and folate-mediated one-carbon metabolism biomarkers in relation to survival, recurrence, treatment toxicity and healthrelated quality of life outcomes in patients with colorectal cancer (CRC). ⇒ The cohorts included in the FOCUS Consortium are designed to enable future pooling of data using harmonised and standardised methods to collect data and biospecimens. ⇒ The pooled sample size provides sufficient power to investigate subgroup analyses across patients with CRC. ⇒ Study time point definitions differ between some of the cohorts and have to be adapted for specific projects. ⇒ A selection bias for follow-up can arise because it is possible that patients who experience more severe toxicities, worse clinical outcome or health-related quality of life are under-represented among those completing follow-ups.

Open access
Many patients with cancer seek information on what they can do themselves to improve survival-for instance, by improving their dietary habits and other lifestyle factors. 5 However, there may be behavioural aspects among individuals diagnosed with CRC, which may be harmful in some cases, such as the use of high-dose nutritional supplements containing synthetic folate. In general, knowledge on short-term and long-term effects is insufficient to make sound recommendations on use of dietary supplements, in particular folate, to cancer survivors, even though dietary supplements are used by 20%-85% of patients with cancer, [6][7][8] highlighting the importance of thorough evaluation of potential benefits and harms and to support development of evidence-based recommendations on use of dietary supplements to patients with cancer. Folic acid is the synthetic form of the B-vitamin folate and is often used in dietary supplements or fortified foods. However, there is broad agreement that food folate is less bioavailable than folic acid with a median relative bioavailability of 65% (range: 44%-80%), an estimate that approximates the 60% value derived from the dietary folate equivalents equation. 9 Folate and folic acid play an important role in one-carbon metabolism, which is a complex series of biochemical reactions essential in nucleotide synthesis, methylation reactions and amino acid homeostasis. 10 One-carbon metabolism refers to a complex network of biochemical reactions linked to nucleotide synthesis and provides methyl groups for DNA, RNA or protein methylation. Thus, one-carbon metabolism is directly controlling processes determining DNA synthesis and integrity, both processes known to be linked to tumour growth. 11 To what extent folic acid supplement use and biomarkers of folate-mediated onecarbon metabolism (FOCM) impact cancer survival and treatment efficacy and toxicity still needs to be clarified. 11 12 Folate and FOCM biomarker deficiencies may increase cancer risk, but high levels, especially of synthetic folic acid, may also be driving factors in carcinogenesis. 6 13 An increasing body of evidence suggests that folate plays a dual role in carcinogenesis, involving both the prevention of early lesions and potential harm once preneoplastic or neoplastic lesions have developed. 6 12 14-20 The overarching goal of the FOCUS (biomarkers related to FOlate-dependent one-carbon metabolism in Colorectal cancer recUrrence and Survival) Consortium is to study associations between folate and FOCM biomarkers and recurrence and survival in patients with CRC. Better insights into these associations will facilitate the development of guidelines regarding folate intake in order to provide clinically relevant advice to cancer patients, health professionals involved in patient care, and ultimately further tertiary prevention strategies in the future. The FOCUS Consortium is a large-scale international consortium with patients with CRC from six prospective cohort studies. The primary objectives of the FOCUS Consortium are as follows: (1) to determine possible associations of folate and other FOCM biomarkers at diagnosis with recurrence or survival in non-metastatic (stages I-III) CRC; (2) to elucidate whether biomarkers related to FOCM are associated with dietary and supplemental intake of these respective nutrients; (3) to explore whether FOCM biomarkers are associated with treatment toxicity in patients with CRC undergoing chemotherapy and (4) to collect comprehensive data of patient characteristics at baseline and follow-up including biomarkers of FOCM to establish an unique resource for future scientific research. The FOCUS Consortium is funded by the European Research Area Network on Translational Cancer Research.
The main purpose of the FOCUS Cohort profile is to (1) inform the scientific community about the FOCUS Consortium, (2) describe the complex methodology of a large consortium, (3) present ongoing studies using this infrastructure and (4) advise interested researchers of opportunities for collaboration. This joint research may lead to a better understanding of the role of folaterelated and FOCM-related mechanisms in the prognosis of CRC and be a precursor for data for future randomised controlled trials (RCTs), which will be critical for the development of guidelines regarding folate intake among patients with CRC.

Cohort description
The FOCUS Consortium is an international, prospective consortium including six cohort studies recruiting women and men at the age of 18 years and older diagnosed with a primary CRC. The FOCUS Consortium is composed of patients from the ColoCare Study at the University of Heidelberg, Germany (n=298, 12 In total, n=2435 patients with CRC were considered for the FOCUS Consortium, of which n=34 patients were excluded due to tumour staging being either 0 or IV leading to a total number of n=2401 stages I-III patients with CRC included in further analyses. Patients were recruited after CRC diagnosis and repeated study measurements were conducted at time of recruitment, and at 6 and 12 months thereafter. At each study time point, sociodemographic data, data on lifestyle factors (eg, diet, supplement use, physical activity) and clinical data (eg, tumour site and stage, cancer treatment, treatment-induced toxicities, recurrence, survival) Open access were collected, and blood samples were drawn (figure 1). Below, a more specific description of each included study is provided.

The ColoCare Study
The ColoCare Study ( ClinicalTrials. gov identifier: NCT02328677) is an ongoing international, multicentre prospective cohort study among women and men newly diagnosed with a primary invasive CRC, with the goal to investigate predictors of cancer recurrence, survival, treatment toxicities and health-related quality of life. 4 21 Three ColoCare Consortium sites participate in the FOCUS Consortium: the Fred Hutchinson Cancer Research Center in Seattle (Washington, USA), and the University Hospital Heidelberg, Heidelberg (Germany) as well as the Huntsman Cancer Institute in Salt Lake City (Utah, USA). Patients are enrolled prior to undergoing CRC surgery according to the following inclusion criteria: individuals who are 18 years of age and older, newly diagnosed (ie, non-recurrent) with invasive CRC. Blood draws and other biospecimens are obtained prior to surgery and at regular intervals (eg, 6, 12, 24 months). Questionnaires are administered to assess lifestyle behaviour, health-related quality of life and clinical outcomes such as CRC recurrence, treatment and treatment symptoms at each study time point. Clinical data are obtained through reviews of patient medical records, pathology and imaging reports. Vital status is obtained through medical records, routine follow-up mailings, periodic requests for outside medical records and state or national cancer and death registries.

The COLON Study
The COLON Study ( ClinicalTrials. gov identifier: NCT03191110) started in 2010 and is an ongoing, multicentre prospective cohort study specifically designed to assess associations between nutrition, lifestyle and dietary supplement use with quality of life, CRC recurrence and survival among patients with CRC (stages I-IV). 22 Persons Open access with a history of CRC or (partial) bowel resection, chronic inflammatory bowel disease, or a known hereditary CRC syndrome are excluded from the study. Patients are recruited from 11 regional and academic hospitals prior to surgery. Individuals donate blood samples and provide information on diet, lifestyle and dietary supplement use at CRC diagnosis, that is, baseline, and at 6, 12 (chemotherapy patients only), 24 and 60 months after diagnosis. Clinical data are collected through review of medical records (treatment-induced toxicity) or through linkage with the Dutch ColoRectal Audit (DCRA). Mortality data (ie, death or alive and date of death) are retrieved from the Municipal Personal Records Database. Recurrence data have been retrieved in collaboration with the Netherlands Cancer Registry.
The CORSA Study CORSA is an ongoing case-control study of women and men recruiting patients with high-risk and low-risk adenomas and population-based colonoscopy negative controls, with an age range between 30 and 90 years. Since 2003, more than 13 500 participants have been recruited across nine sites in Austria. The multicentre recruitment within CORSA follows standardised protocols, resulting in consistent data from all recruitment sites. These sites include the Medical University of Vienna (Department of Surgery), two hospitals in Vienna (Clinic Favoriten and Clinic Landstraße) and the Hospital Wiener Neustadt in Lower Austria. Furthermore, the recruitment for CORSA was performed in four hospitals in the federal state Burgenland within the population-based screening programme 'Burgenland PREvention Trial of colorectal cancer DIsease with ImmunologiCal Testing' (B-PRE-DICT). B-PREDICT is a two-stage screening project where more than 150 000 inhabitants of Burgenland aged between 40 and 80 are invited annually to participate in this programme using a faecal immunochemical test (FIT) as an initial screening. FIT-positive (≥10 µg haemoglobin/g faeces) tested individuals are offered a complete colonoscopy and are asked to participate in CORSA. Biospecimen are collected at each site using harmonised protocols. CORSA participants provide a basic CORSA questionnaire assessing data on body mass index (BMI), smoking history, alcohol consumption, education level, family status, profession, basic dietary habits and diabetes. Clinical data are abstracted from medical records and processed in a structured database following standardised documentation guidelines and according to the General Data Protection Regulation.

The EnCoRe Study
The EnCoRe Study is a prospective cohort study initiated in 2012 at the Maastricht University Medical Center+in the Netherlands that focuses on the importance of lifestyle factors for quality of life, recurrence and survival of patients with CRC. 23 The EnCoRe Study is registered in the Netherlands Trial Registry for experimental and observational studies (www.trialregister. nl, registration number 7099). Stages I-III patients with CRC are enrolled at diagnosis at three hospitals in the South-Eastern region of the Netherlands and followed up until 5 years after completion of treatment with repeated measurements at diagnosis (pretreatment), and at 6 weeks and 6, 12, 24 and 60 months after the end of the initial anticancer treatment (ie, surgery, chemotherapy, radiotherapy). Patients with stage IV CRC, an inability to understand the Dutch language in speech or writing, with comorbidities obstructing successful participation (eg, Alzheimer disease), or with severe visibility or hearing disorders are excluded from the study. Repeated home visits by trained dieticians are conducted and data are collected among others on sociodemographic factors, quality of life, functioning, physical activity, comorbidity, dietary intake, supplement use and anthropometry. In addition, clinical data are collected from hospital records and blood samples are drawn at all time points. Recurrence data have been retrieved in collaboration with the Netherlands Cancer Registry. Mortality data (ie, death or alive and date of death) are retrieved from the Municipal Personal Records Database.

The FOCUS Consortium
Sociodemographic and clinical characteristics of the 2401 participants in the FOCUS Consortium are presented in table 1. The mean age at CRC diagnosis was 65.4 years (SD: 10.2; range: 22-93 years), and the majority of participants were male (64.0%). The mean BMI was 27.1 kg/ m² (SD: 4.6).
In terms of education status, 35.9% of the patients reported lower education, 28.9% reported intermediate education and 27.4% high education. Most of the patients were married or part of a living community (73.2%). About half of the overall cohort reported to be a former smoker (50.8%), 31.9% were never-smokers and 12.1% were current smokers (5.2% were unknown). Mean alcohol intake was 13.6 g/day (SD: 17.1). In total, 16.9% of the patients reported regular dietary supplementation (ie, at least once per week during the last 4 weeks) of folic acid. Over half of all participants (54.5%) reported adherence to the physical activity guidelines of at least 150 min per week of moderate-to-vigorous physical activity. Lifestyle characteristics of the FOCUS cohort are presented in table 2.
Regarding clinical characteristics, 60.4% of participants were diagnosed with colon cancer, 35.9% with rectal cancer, and 1.7% with rectosigmoid cancer (2.0% were of unknown tumour subsite). In total, 26.2% were diagnosed with stage I, 28.9% with stage II and 40.5% with stage III CRC. Approximately 1% of participants were classified with an unspecified cancer stage, as distinction between stage I and II or II and III was not possible, and for 3.7% of the total population the cancer stage was unknown. In total, 95.4% of patients underwent surgery, whereas neoadjuvant chemotherapy was administered to 13.8% of patients and 28.4% received adjuvant chemotherapy. Neoadjuvant radiotherapy was administered to Open access Open access 21.8% of patients and adjuvant radiotherapy in 9.5% of the population.

Patient and public involvement
Patients and the public were not involved in the design, conduct, reporting or dissemination plans of this research.

Cohort follow-up
At each study site, collection of biospecimen, clinical, demographic, questionnaire and anthropometric data occurred at baseline, and at 6 and 12 months following recruitment. Baseline measurements in CORSA, COLON and EnCoRe have been performed at diagnosis (preferably prior to any cancer treatment) while in the Colo-Care Study such measurements have been done prior to surgery (ie, neoadjuvant treatment might have been applied prior to baseline blood and data collection). Blood samples at baseline were collected from n=2132 (88.8%) study participants. N=1537 (64.0%) participants donated blood at the 6 months and n=614 (25.6%) at the 12 months follow-up time point. In addition, n=251 (10.5%) EnCoRe patients provided blood samples at 6 weeks post-treatment, a subset specifically of interest at the Maastricht study site.

Data collection
Data collection at baseline and follow-up time points is summarised in table 3 and is briefly described below.
Lifestyle and demographic data ColoCare questionnaires and standardised Food Frequency Questionnaires (FFQ) are used to assess intake of dietary supplements and medication, smoking, dietary intake and other health behaviours at each study time point. In COLON, patients provide information on diet, lifestyle and supplement use via COLON questionnaires and FFQs. Patients from the CORSA Study are requested to complete a questionnaire assessing anthropometric and demographic factors. Patients enrolled in EnCoRe receive repeated home visits by trained dieticians, where extensive measurements are performed that include assessment of demographic data, physical activity (both questionnaire and accelerometry based) and smoking behaviour (questionnaire data), dietary intake (FFQs at diagnosis and 7-day food diaries at follow-up measurements), supplement use (registered by dieticians) and anthropometry measurements.
Clinical data and outcomes: medical chart abstraction All cancers and medical diagnoses are classified according to International Classification of Diseases, 10th Revision codes. Details on CRC treatment, including type of treatment regimens and treatment toxicity, are abstracted via medical records for all cohorts. Detailed information on the primary outcomes of interest, CRC recurrence and survival, are ascertained through reviews of medical records, pathology reports and imaging reports documenting the diagnosis of a recurrence. Data on recurrence and vital status is supplemented from the clinical cancer registries and survival data are verified by the inhabitant registries that exist at all study sites. For COLON and EnCoRe, all data on recurrence are retrieved in collaboration with the Netherlands Cancer Registry. Moreover, survival data for CORSA participants were obtained by the Main Association of Austrian Social Insurance Carriers as well as from Statistics Austria.

Patient-reported outcomes
Health-related quality of life is assessed by the validated and widely used cancer-specific 30-item core questionnaire QLQ-C30 and the 29-item CRC module QLQ-CR29, developed by the European Organisation for Research and Treatment of Cancer (EORTC). 24 Chemotherapyinduced peripheral neuropathy (CIPN), which is a common toxicity in patients with CRC, is measured by the EORTC QLQ-CIPN20. 25 Patient-reported outcomes are available for ColoCare Heidelberg, COLON and EnCoRe.

Biomarkers of FOCM
Blood is processed in identical settings across all study centres. Plasma (CORSA, COLON and EnCoRe) and serum (for the three ColoCare Study sites) samples were collected and immediately centrifuged, aliquoted and stored at −80°C.
All biological analyses were performed at BEVITAL AS (Bergen, Norway, http://www.bevital.no), which carried out metabolic profiling of biomarkers allocated to seven complementary analytical platforms. Apart from analyses of microbiological active folate 26   218 (100) 6 (1.9) *Self-reported engagement in at least 150 min per week of moderate-to-vigorous physical activity.  30 were analysed by liquid chromatography-tandem mass spectroscopy, whereas other amino acids, in addition to total homocysteine, total cysteine and methylmalonic acid were analysed by gass chromatography-tandem mass spectroscopy. 31 hsCRP and cystatin C and its variants were measured by MALDI-TOF mass spectroscopy. 32 A comprehensive overview of the panel of biomarkers measured in blood samples of patients enrolled in the FOCUS Consortium is provided in table 4.

Findings to date
The FOCUS Consortium provides a unique opportunity to conduct comprehensive research on folate and FOCM   Open access biomarkers status in the tertiary prevention of CRC using data collected both at diagnosis and during standardised follow-up time points. This well-characterised study design provides sufficient statistical power to discern prospective associations with relevant clinical outcomes, including CRC recurrence and survival, within relevant subgroups.

Key findings and publications
We investigated associations of circulating concentrations of folate, folic acid and folate catabolites pABG and apABG, measured around time of diagnosis, with recurrence and survival among 2024 patients diagnosed with stages I-III CRC within the international FOCUS Consortium. We did not observe any statistically significant associations for folate, pABG and apABG concentrations. However, an increased risk of cancer recurrences was observed among patients with higher compared with lower circulating folic acid concentrations. 33 Further, Kiblawi et al measured associations between one-carbon metabolites, inflammation-related and angiogenesisrelated biomarkers in a cross-sectional analysis of 238 patients from the ColoCare Heidelberg cohort. The study showed that specific folate species within the one-carbon metabolism pathway are associated with both inflammation and angiogenesis pathways among patients with CRC. In particular, vitamin B 6 species, pyridoxal 5'-phosphate (PLP), pyridoxal (PL) and pyridoxic acid (PA), were Folate and one-carbon metabolites Abbreviation Description

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inversely associated with inflammatory biomarkers CRP, serum amyloid A, IL-6 and IL-8. Thiamine and thiamine monophosphate were inversely correlated with the CRP and IL-6. In addition, positive correlations of PA, PL and PLP with angiogenesis biomarker VEGF-D were observed. Our findings reinforce the notion that B vitamins involved in the one-carbon metabolism may be correlated with carcinogenic processes. 34 This and further research will support the evidence base needed for the development of dietary guidelines for patients with CRC. Further, we investigated circulating concentrations of nine biomarkers related to the B-vitamins folate, riboflavin, vitamin B 6 and cobalamin, measured at diagnosis and 6 months postdiagnosis, in association with healthrelated quality of life as assessed by the EORTC QLQ-C30 questionnaire 6 months post-treatment in three FOCUS cohorts (ColoCare Heidelberg, EnCoRe and COLON). 35 Higher PLP concentrations were cross-sectionally associated with better physical, role and social functioning, and reduced fatigue 6 months postdiagnosis. Higher HKr (3-hydroxykynurenine:(kynurenic acid+xanthurenic acid+3-hydroxyanthranilic acid+anthranilic acid)), an inverse marker of vitamin B 6 status, was cross-sectionally associated with worse global quality of life, and lower physical and role functioning. Dose-response relations were observed for PLP with global quality of life, physical, role and social functioning. No associations were observed for changes in biomarker concentrations between diagnosis and 6 months with quality of life outcomes. We, therefore, concluded that higher vitamin B 6 status was associated with better quality of life at 6 months post-treatment and that further study is needed to clarify the role of vitamin B 6 in relation to quality of life.
Previous relevant findings from individual cohorts within the consortium To date, individual cohorts from the FOCUS Consortium have initiated the examination of dietary supplement use and dietary habits over time. Among patients with CRC enrolled in the ColoCare Study the proportion of supplement users was found to be highest post-diagnosis (35%). 36 Moreover, within an international investigation including ColoCare participants from multiple sites, Ulrich et al showed differences in plasma folate concentration between Heidelberg and the US sites, probably reflecting variation in folic acid fortification and supplement use. 13 Furthermore, ColoCare has published data on RECQ helicase expression, 37 NTRK3, 38 RET, 39 tumourinfiltrating lymphocytes and T cell receptor sequences, 40 25-25-hydroxyvitamin D 3 , 13 41 DNA methylation, [42][43][44][45] miRNAs, 46 47 faecal microbiota, 48-50 metabolomics and transcriptomics, 51-53 plasma proteins, 54 gene expression, 55 branched-chain amino acids, 56 genetic variants, 57 body composition, 53 58 59 physical activity 41 and dietary patterns 4 in patients with CRC. Within the COLON study, results have been published on body weight trajectories, 60 changes in lifestyle, 61 25-hydroxy vitamin D levels, 62 63 and inflammation markers 64 over time, as well as vitamin D, 64 calcium or magnesium intake, 65 physical activity, 66 inflammation, 67 68 skeletal muscle mass 69 /density 70 and other measures of body composition 71 in relation to cancer recurrence, survival or physical functioning or fatigue. Moreover, in a subset of patients undergoing chemotherapy, dietary factors in relation to chronic CIPN 72 73 as well as chemosensory perception and food preferences 74 were studied. The Austrian CORSA Study has published results on genomic data, [75][76][77] telomere length, 78 DNA repair processes, 79 tumour autoantibodies 80 as well as metabolomics. 33 81 To date, publications from the EnCoRe Study have reported on associations of physical activity and sedentary behaviour, [82][83][84][85] adherence to lifestyle guidelines, 86 and parameters of body composition 87 88 measured through CT scans with quality of life, functioning and fatigue in CRC survivors. Recently, longitudinal associations between supplement use and fatigue were investigated from diagnosis to 2 years post-CRC treatment. No overall association between supplement use and fatigue was found but results suggest that increased levels of fatigue may be a reason for the use of supplements among CRC survivors. 89 Higher concentrations of 25OHD3 were longitudinally associated with better global quality of life and less fatigue in CRC survivors within EnCoRe suggesting a potential beneficial role of vitamin D in colorectal cancer survivors. 35 In a mixed-method study using data of the EnCoRe study, CRC (treatment) related health and functioning problems negatively impacted the ability of nearly one in five long-term CRC survivors to participate in everyday life situations and their satisfaction with participation. 90 The validity of the FFQ for measuring dietary intake among survivors of CRC within the EnCoRe study appeared to be moderate to good for most nutrients and food groups, relative to a 7-day dietary record. 91 Prediction models were developed for estimating 1-year risk of low health-related quality of life in seven domains in CRC survivors and performed well when externally validated among survivors within the EnCoRe and COLON studies. 92

Future plans
The consortium specified a comprehensive manuscript list of future projects using data from the FOCUS Consortium. Some selected projects are described below: a. Recently, the investigation of longitudinal associations of adherence to the dietary World Cancer Research Fund/American Institute for Cancer Research and Dutch Healthy Diet recommendations with plasma kynurenine levels in CRC survivors after treatment has been finalised and the corresponding manuscript is under review at an international journal. b. Further, near-term future plans include the investigation of (1) biomarkers related to FOCM and associations with folate intake (from diet and supplements); (2) associations between FOCM biomarkers such as vitamin B 12 and tryptophan and recurrence, survival, and patient-reported outcomes in CRC; (3) the impact of folate status (FOCM biomarkers and diet/ Open access supplements) on treatment toxicity in patients treated with 5-floururacil modifiers; (4) the interaction between biomarkers related to FOCM and polymorphisms in FOCM-related genes in relation to CRC prognosis (recurrence and survival); (5) prognosis (disease-free and overall survival) in stages I-III CRC and associations with dietary and supplement use at diagnosis and changes during and after treatment; (6) FOCM-related biomarkers and their association with body composition in stages I-III patients with CRC; (7) associations between folate status (FOCM biomarkers and diet/supplement use) and recurrence, survival and patient-reported outcomes in young-onset CRC. c. Long-term plans include the combination of additional biomarkers measured by the individual cohorts within the next years (eg, microbiome data, omics and multiomics data). therefore also be limited. 93 Performing sensitivity analyses by excluding countries without folic acid fortification (eg, Germany) or investigating analyses separately for Germany and the USA might help to address differences in fortification status. Moreover, patients were predominantly White, thus, it is not possible to address racial and ethnic minorities. Ethnicity/race is an important determinant of folate status and metabolism may be different between African Americans and Hispanics, 94 thus, recommendations should be limited to this current population. Future studies are warranted in diverse populations and compared with the FOCUS cohort. Another limitation of the consortium includes differences in collection strategies of the baseline study time point across included studies: CORSA, COLON and EnCoRe are recruiting patients with CRC at diagnosis, preferably prior to any cancer treatment while ColoCare is recruiting patients prior to surgery (ie, neoadjuvant treatment might have occurred prior to baseline blood and data collection). Nevertheless, the ColoCare protocol requires that no blood draw occurs within 2 weeks of neoadjuvant or adjuvant chemotherapy, limiting the influence of ongoing treatments on blood biomarkers. In some cohorts, the timing with respect to adjuvant chemotherapy is less clearly defined and this will be carefully considered in sensitivity analyses. Furthermore, survivor bias-a type of selection bias-may be introduced as some patients died or did not complete follow-up questionnaires or did not provide blood samples. It is possible that patients who experience more severe toxicities, worse clinical outcomes, or worse health-related quality of life, are underrepresented among those patients who completed follow-up measurements. 4 Cohort studies such as the one presented here generate critical knowledge about preventable causes of disease. However, selection bias may affect estimates. This is particularly true for non-participation at follow-up that may depend on both the exposure and outcome. Within a review, Nohr et al showed a range of methods to quantify and adjust for selection bias. Even with limited data on nonparticipants and those lost to follow-up, it is possible to examine how effect estimates in a specific study may be biased by selection. 95 The likelihood for reverse causation is small in this prospective cohort, as the exposure measurements (blood folate levels and intake through diet/supplements) were collected before the outcome (survival, recurrence and quality of life) occurred. Therefore, these outcomes are unlikely to have influenced the exposure measurements. Given the robust follow-up in these cohorts for outcomes and data availability, future studies will be able to consider key confounders as well as predictors of recurrence and survival.

Strengths and limitations
Open access from European-based consortia and as such are subject to regulations from multiple European countries, which limit our availability to share data. The consortium's funding has ended, and no centralised staff is available to support data requests. However, the FOCUS PIs have agreed to answer any queries or discuss potential projects with anyone interested in future collaborative research. For further questions, please contact colocarestudy_ admin@ hci. utah. edu.
Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/.